Progression of mammary carcinogenesis involves numerous alterations in signaling pathways and transcription factors. We are studying two types of nuclear receptor ligands, a novel vitamin D receptor ligand (a non- hypercalcemic Gemini vitamin D analog) and a PPARg ligand (a unique triterpenoid), for inhibition of breast cancer progression. Our preliminary data show that Gemini vitamin D analogs and triterpenoids regulate the Smad signaling pathway and synergize to inhibit the proliferation of cultured MCF10 breast cancer cells. We hypothesize that Gemini vitamin D analogs and triterpenoids regulate nuclear receptor signaling and the TGF- b/Smad system, and these effects contribute to their inhibition of the progression of breast cancer in vivo. To test this hypothesis, we plan to pursue the following Specific Aims: (1) investigate the molecular mechanism of a representative Gemini vitamin D analog and a triterpenoid, alone and in combination, in cultured MCF10 breast cancer cell lines (with different degrees of progression), (2) determine the in vivo efficacy of a Gemini vitamin D analog and a triterpenoid in an animal model of MCF10 breast cancer and elucidate the mechanisms of action involved, and (3) verify the in vivo efficacy of the Gemini vitamin D analog and a triterpenoid in an animal model of MMTV-neu estrogen receptor (ER)-negative breast cancer. To study the in vivo efficacy and molecular mechanisms of these agents against breast cancer progression, we will use the MCF10 cell lines that represent different stages of progression when injected into immunodeficient mice. We will investigate the molecular mechanism of synergy of these agents by examining (a) interactions of nuclear receptors and coactivators, (b) regulation of transcriptional activation and target genes, and (c) regulation of Smad signaling and post-translational modification by a Gemini vitamin D analog and a triterpenoid in the MCF10 breast cell lines. The MCF10 breast cancer xenograft model and the MMTV-neu breast cancer transgenic mouse model will be used for determining in vivo efficacy of a Gemini vitamin D analog and a triterpenoid, alone and in combination, in ER-negative breast cancer. Our proposal focuses on the identification of novel nuclear receptor ligands as promising agents for the inhibition of ER-negative breast cancer. This is important because there are currently only a limited number of agents for the prevention/treatment of ER-negative breast cancer. Our research with a novel non-hypercalcemic Gemini vitamin D analog and a novel triterpenoid that inhibits breast cancer progression will provide important preclinical data for the prevention of ER-negative breast cancer.Project Summary/Narrative This proposal is to study two nuclear receptor ligands, a novel non-hypercalcemic Gemini vitamin D analog and a novel triterpenoid, as agents for inhibition of breast cancer formation and progression. We will investigate the regulation of nuclear receptor signaling with a focus on the TGF-b/Smad signaling pathway by these two agents, and we will determine their in vivo efficacy in animal models of estrogen receptor negative breast cancer progression. We anticipate finding synergistic inhibition of breast cancer progression by the combination of a Gemini vitamin D analog and a unique triterpenoid. This research will be an important precursor for a clinical cancer prevention trial in patients with a high risk of estrogen receptor negative breast cancer.